The long-term objective of this research is to develop highly directed and long-lasting therapy for rheumatoid arthritis (RA). The specific aims are to, for the first time: 1) Study physical associations between heat shock protein (HSP70) and citrullinated-fibrinogen 2) Demonstrate mechanism of delivery of cit-fibrinogen into immature dendritic cells (DC). The recognition that cit-proteins (especially cit-fibrinogen) are key auto-antigens (Ags) initiating and perpetuating immunopathology in RA may represent a huge step toward achieving more effective therapy. To date, however, many of the events related to the handling of cit-Ags are unknown, including mechanisms of cit-autoAg uptake/processing by antigen presenting cells such as DC. The hypothesis is that in RA, citrullinated Ags such as cit-fibrinogen are transported via HSP into immature DC to activate autoimmune lymphocyte responses. In RA, HSP are highly abundant and likely to facilitate the transport of auto-Ags into immature DC. Because citrullination of proteins results in unfolding and partial denaturing, citproteins are prime candidates for association with HSP, which partner with proteins in non-native states. Emphasis will be placed on studying inducible HSP70 and cit-fibrin, which are specifically elevated in RA synovial fluid (SF) vs non-RA SF and considered key self-cit-Ags in RA, resp. In Aim 1, HSP70-cit-fibrinogen interactions will be studied using surface plasmon resonance (SPR) and co-immunoprecipitation (IP) techniques. For SPR, purified proteins will be used. HSP70-cit-fibinogen complexes present in RA SF, RA serum, non-RA SF and normal serum will be revealed by IP. In Aim 2, immature DC will be primed in vitro to undergo an inflammatory/heat stress response under precise conditions present in the RA joint. After the addition of cit-fibrinogen, uptake/intracellular trafficking of cit-fibinogen will be assessed by IP and confocal microscopy. Due to the increased affinity of cit-Ags to HLA-DR4, studies of cit-fibrinogen-HLA-DR interactions and cit-fibrinogen uptake in DC prepared from RA patients expressing the common epitope will be included. These studies will employ synthetic cit-fibrinogen 10mer peptides (containing cit-epitopes) and native fib peptides and purified HLA-DR4 and HLA-DR1 (non RA linked). Understanding how key self-antigens in RA are handled by the immune system at the earliest stages of the autoimmune process may reveal a universal disease mechanism that can be specifically interrupted, before the onset of severe disease activity.